Electrospray Ionization (ESI) of solute species in a volatile liquid solvent is carried out by dispersing the liquid as a fine spray of highly charged droplets in a bath gas. As solvent evaporation shrinks the droplets they pass through a somewhat intricate sequence of steps that leads ultimately to the transformation of polar solute species in the droplet liquid to free ions in the ambient bath gas. Some of the resulting ion-gas mixture can be admitted into a vacuum system where the ions can be "weighed" by a mass analyzer. This combination of ESI with mass analysis in so-called Electrospray Ionization Mass Spectrometry (ESIMS) can produce and weigh intact ions from simple polar molecules as well as from complex and fragile species with molecular weights up to many millions. The ESIM ions of large molecules are multiply charged so their mass/charge (m/z) ratios are low enough for weighing by relatively inexpensive instruments such as quadrupole mass filters and ion traps. Sensitivity is so high that a complete analysis may require only attomols of analyte. These features of the ESIMS technique have brought about an explosive expansion in its use. In the archival journals of 1984 there were only two papers on the subject [M. Yamashita and J. B. Fenn, Journal of Physical Chemistry 88, 4451 and 4471 (1984)]. In 1996 alone there were around 800 papers relating to the mechanisms, procedures and applications of ESIMS. The world population of ESIMS systems, now around 5000, is expected to grow rapidly as they increasingly become the detector of choice for liquid chromatographs of which around 12,000 are sold annually.
To provide some background perspective for the present invention we present a brief operational description of the ESIMS method along with some examples of results. FIG. 1 shows a schematic diagram of an ESIMS apparatus similar to that described in US patents of Labowsky et al (4,531,056) and Yamashita et al (4,542,293). It also resembles the systems described in US Patent of Henion et al (4,861,988) and Smith et al, (4,842,701 and 4,885,706) as well as in review articles [Fenn et al, Science 246, 64 (1989); Fenn et al, Mass Spectrometry Reviews 6, 37 (1990); Smith et al, Analytical Chemistry 2, 882 (1990)]. It will be useful to set forth the essential features of the technique with reference to FIG. 1. Sample solution at a few microliters/minute (uL/min) is injected through hypodermic needle 1 into an opposing flow of bath or drying gas 2 (e.g. a few L/min of warm dry nitrogen) in electrospray chamber 3 whose walls serve as a cylindrical electrode and whose pressure is typically maintained at or near one atmosphere. In the end wall of chamber 3 is glass capillary tube 4 with typical dimensions in mm of: L=180, OD=6, and ID=0.6. The front face of glass capillary tube 4 is metalized and held at a few kV "below" the potential of injection needle 1 which can be at any desired potential including ground. Cylindrical electrode (spray chamber 3) is at a potential intermediate between that of injection needle 1 and metallized face of glass tube 4. The resulting electric field at the tip of needle 1 disperses the emerging liquid into a fine spray of charged droplets. Driven by the field the droplets drift toward the inlet of tube 4, shrinking as they evaporate solvent into the opposing flow of drying gas 2. This shrinking increases each droplet's surface charge density until the so-called Rayleigh limit is reached at which electrostatic repulsion overcomes surface tension and a "Coulomb explosion" disperses the droplet into a plurality of smaller droplets which repeat the sequence of evaporation and explosion. Then the droplets become small enough a charge density below the Rayleigh limit can produce an electric field normal to the droplet surface that is strong enough to evaporate or desorb solute surface ions into the ambient bath gas. This Ion Desorption Mechanism, proposed by Iribarne and Thomson [J. Chem. Phys. 64, 2287 (1976) and 71, 4451 (1979)] is now accepted by many investigators. Others favor a Charged Residue Mechanism (CRM) proposed by Malcolm Dole and his colleagues [J. Chem. Phys. 49, 2240 (1968) and 52, 4977 (1970)]. It assumes that the evaporation-explosion sequence leads to ultimate droplets so small that each one contains only a single solute molecule that becomes an ion by retaining some of that ultimate droplet's charge as the last solvent evaporates.
By whatever mechanism they may be formed, the ions along with the evaporating droplets drift down the field, counter-current to the flow of drying gas to arrive at the entrance of glass tube 4 where some are entrained in dry bath gas that emerges into first stage 5 of a vacuum system as a supersonic free jet. A core portion of that jet passes through skimmer 6 and electrostatic lens stack: 7 delivering ions to mass analyzer 8 in second vacuum stage 9. The ions entering glass tube 4 are in a potential well whose depth is the difference in voltage between needle 1 and the entrance of the glass tube 4. The flow of gas through the glass tube drags the ions up out of said well to any desired potential at the tube exit, even many KV above ground! By this arrangement all external parts of the apparatus are at ground potential, posing no hazard to an operator.
In the system of FIG. 1 just described the counter-current flow of warm bath gas achieves evaporation of droplet solvent and desolvation of the resulting ions before they enter the glass tube leading into the vacuum system containing a mass analyzer. However, there are some variations on this general approach which can also deliver desolvated ES ions to the mass analyzer. Some systems avoid the need for counter-current gas flow and achieve most of the desolvation of droplets and ions by raising the temperature of the mixture of droplets, ions, and bath gas, or a portion thereof, before it enters the vacuum system. One such system passes a portion of said mixture of ions and solvent-containing bath gas through a heated metal tube instead of glass tube 4 of FIG. 1. The metal tube walls are sufficiently hot to raise the gas temperature enough to avoid resolvation of the ions due to adiabatic cooling during the free jet expansion of the ion-bearing bath gas at the exit of the tube by which the ions and bath gas enter the vacuum system. Any residual solvation of the ions can then be eliminated by maintaining a voltage difference between the tube exit and the skimmer. The resulting potential gradient accelerates the ions relative to the neutral bath gas molecules during the free jet expansion, thereby bringing about ion-neutral collisions sufficiently energetic to strip the ions of any remaining solvent molecules. Such a system was originally proposed by Chowdhury, Katta and Chait et al and is described in U.S. Pat. No. 4,977,320 as well as in a paper. [Rapid Communications in Mass Spectrometry, 81 (1990)]. If the conduit for passage of ions and bath gas from the ES chamber into the vacuum is made of a conducting material such as a metal, then there cannot be an appreciable potential difference between the inlet and the exit ends of the conduit. Therefore, the ES injection needle for sample liquid must be maintained at a potential substantially above ground in order to provide a field at the needle tip that is intense enough to electrospray the emerging liquid. In other words the source of sample liquid must itself be floating above ground or else provision must be made to raise the sample liquid from ground to the needle potential which may be several KV above ground. This requirement can introduce some design problems when, the source of sample liquid is a liquid chromatograph.
Some systems have only one vacuum stage which is provided with enough pumping speed to accommodate the entire flow of gas from the electrospray region while maintaining the background pressure low enough for satisfactory operation of the mass analyzer. In one such single stage system much of the ion desolvation is achieved by means of a potential gradient in the jet as described above. Still other systems incorporate one or more additional stages of pumping between the first vacuum stage (5 in FIG. 1) and the final vacuum stage (9 in FIG. 1) containing the mass analyzer (8 in FIG. 1). In some systems the conduit through which ion-bearing bath gas passes from the electrospray region into the vacuum system is a simple orifice instead of a tube. Common to all systems is the electrospray region or chamber in which the sample solution is dispersed into a bath gas at a pressure high enough so that the mean free path is small relative to the diameter of the exit aperture. In other words the bath gas must be dense enough to provide the enthalpy required to evaporate solvent from the ES droplets and to slow down ion mobility enough to prevent space charge from dispersing the ES ions to such a low concentration in the bath gas entering the vacuum system that the mass analyzer can't provide useful signals. At present the pressure of bath gas in most ESIMS systems is at or near one atmosphere but some investigators have been exploring the possibility of carrying out ESI at much lower pressures. The present invention relates primarily to what goes on in the electrospray chamber before the ion-bearing gas enters the conduit into the vacuum system. Consequently, it should be equally applicable in most ESIMS systems no matter what configuration is used downstream of the ES chamber.
ES ions are believed to be formed from the excess cations (or anions) on a droplet that give rise to its net charge and thus to the current carried by the spray. Those excess cations or anions are believed to be distributed on the surface of their source droplets. They may be alone or in aggregation with normally neutral solute or solvent molecules containing one or more polar groups to which an excess anion or cation can be bound by forces due to charge-induced dipoles, hydrogen bonds, or dispersion. It is these surface ions or ion-neutral aggregates that according to the Ion Evaporation Model (IEM) are desorbed by the droplet's surface field into the ambient gas. FIG. 2 shows some examples of early ESIMS spectra obtained with the apparatus of FIG. 1. Panel 2A is the mass spectrum for a mixture of tetra alkyl ammonium or phosphonium halides at concentrations from 2 to 10 ppm in aqueous methanol. This spectrum was the first proof that ESI could make ions from species that cannot be vaporized without catastrophic decomposition. The spectral peaks of FIG. 2-B are for the decapeptide gramicidin S whose basic groups attach solute protons. It is noteworthy that the dominant peak is for ions with two charges rather than one.
FIG. 2-C shows an ESMS spectrum for the protein cytochrome-c, typical of spectra for molecules large enough to require multiple charges for "lift off " by the droplet field. The charges on ES ions of proteins and peptides are usually protons. The ions of each of the multiple peaks differ from those of adjacent peaks by a single charge (proton). Because of this coherence each peak becomes in effect an independent measure of the molecular weight Mr of the parent species. Mann, Meng and Fenn [U.S. Pat. No. 5,130,538; Analytical Chemistry 61, 1702 (1989)] introduced computer algorithms that can integrate and average the contributions from each peak to arrive at a most probable value of Mr, more accurate and reliable than can be achieved for large molecules by most other techniques. The inset in FIG. 2-C shows the result of such a deconvolution for the illustrated spectrum. The ordinate scale is the same for both the deconvoluted spectrum in the inset and the original experimental spectrum. Clearly, both the effective "signal" and the signal/noise ratio are substantially higher in the deconvoluted spectrum.
Microscopic examination of a stable spray shows that the liquid emerging from the tip of the spray needle forms a conical meniscus known as a Taylor cone in honor of G. I. Taylor whose theoretical analysis predicted that a dielectric liquid in a high electric field would take such a shape. In the case of conducting liquids a fine filament or jet of liquid emerges from the cone tip and breaks up into nearly monodisperse droplets whose initial diameters are slightly larger than the diameter of the jet. Sprays produced under these circumstances are sometimes referred to as "cone-jet sprays." It turns out that to obtain a stable cone-jet electrospray which produces a high yield of solute ions one must achieve an optimum balance between flow rate and applied field. Moreover that optimum balance depends very strongly on the properties of the sample liquid, in particular its electrical conductivity and its surface tension. In general, the higher the conductivity and surface tension, the lower must be the flow rate. In most ESI systems the sample liquid enters the needle at a fixed flow rate determined by a positive displacement pump. In some cases it is convenient to achieve such a fixed flow rate by pressurizing a reservoir of the sample liquid with gas. The liquid flows through a conduit long enough and narrow enough to require a high pressure difference between the source and the exit of the spray needle to maintain the flow. If that pressure difference is very high relative to the pressure at the needle exit, minor pressure fluctuations at the needle tip or in the ES chamber will not affect the liquid flow rate. Thus a steady flow can be maintained at a desired value by appropriate choice of reservoir gas pressure. If the liquid flow rate is higher than the rate at which the electric field can extract liquid from the tip of the Taylor cone, excess liquid accumulates at the base of the cone and periodically departs as a large droplet that interrupts the cone and the spray until enough liquid accumulates to re-establish the cone-jet spray, whereupon the same interruption recurs. If the liquid flow rate is too small the electric field extracts liquid from the tip of the Taylor cone faster than new liquid comes into the base. Therefore, the cone liquid is depleted and the spray stops until enough new liquid accumulates to start again. Even for solutions of analytical interest that are readily sprayable, i.e. have values of surface tension and conductivity that are not too high, it becomes increasingly difficult to obtain stable sprays at flow rates much above about 20 uL/min or so. Best results are generally obtained at flow rates below about 10 uL/min. The maximum flow rate for spray stability depends strongly upon the electrical conductivity of the liquid. The higher the conductivity, the lower is the maximum flow rate for a stable spray.
It has generally been found over a wide range of liquid flow rates that analytical sensitivity increases as liquid flow rate decreases. That is to say the lower the flow rate of a particular sample liquid into the spray, the higher will be the mass-selected ion currents for analyte species in that liquid and/or the higher will be the fraction of analyte species in the liquid that is converted into free ions. Because lower flow rates result in a lower consumption of sample liquid, the overall analytical sensitivity is generally much higher at low flow rates. The reasons for this effect are not entirely clear but are probably due to the decrease in droplet size that results with decreasing flow rate. Small droplets evaporate more quickly and completely than large ones. Moreover, the spray current does not decrease linearly with flow rate so that droplet charge/mass ratio goes up as flow rate, and therefore droplet size, go down.
These restrictions on flow rate, surface tension and conductivity along with the difficulties in achieving and maintaining the right balance between them, constitute substantial handicaps in many applications for which the many advantages of ESIMS are much to be desired. For example, one of the most attractive and important of these applications is as an interface between a Liquid Chromatograph and a Mass Spectrometer in what is commonly referred to as LCMS analysis. The ability of mass spectrometry to identify the species forming a peak in the effluent from a liquid chromatograph has greatly expanded the scope and power of both techniques. Unfortunately, standard practice in LC has long been based on column flow rates of one mL/min whereas stable electrosprays are readily obtainable only with flow rates less than 20 or so uL/min. In recent years LC at much lower flow rates has been increasingly practiced but many large scale users of LC, e.g. the pharmaceutical industry, have very large investments in equipment and protocols based on LC at one mL/min flow rate. These LC users do not welcome the prospect of writing off that investment and undertaking the formidable task of investing in new equipment and developing protocols that will work at flow rates of a few uL/min. Moreover, when one goes to very low flow rates in LC the tolerance of the equipment and the process for impurities and dirt decreases markedly. For these and other reasons it seems likely that LC at relatively high flow rates is likely to be practiced for a long time. Another problem in LC-ESIMS is that the mobile phase in LC is frequently not readily sprayable because of high conductivity and/or surface tension. For all these reasons, and others, there have been substantial efforts on the part of many investigators to make ESI work with flow rates, surface tensions and conductivities that are much higher than it likes.
An effective approach to the problems caused by high surface tension and conductivity in the sample liquid was introduced by R. D. Smith and H. R. Udseth [U.S. Pat. No. 4,885,076]. They provided a small annular or "sheath" flow of an "ES friendly" liquid around the flow of sample liquid emerging from the injection needle. For example, it is very difficult to obtain a stable electrospray with high conductivity solution of analyte in water, especially at a flow rate of five or ten uL/min. However, a stable spray is readily obtained if one provides a small sheath flow of methanol, ethanol or other sprayable liquid.
The problem of obtaining stable sprays at high flow rates is not so easily solved. One almost obvious possible solution to this problem is to divide the effluent flow from the LC into a small stream and a large one. The small stream comprises a flow rate of a few uL/min or less, suitable for ES dispersion to produce ions for mass analysis. The large stream, comprising the remainder of the LC effluent, can be simply discarded or diverted to an autosampler for collection of the fractions (peak species) that might be used for other purposes. Such flow splitting of a primary flow of one ML/min is not hard to achieve when the minor fraction is a few tens of uL/min or more but becomes much more difficult when the minor fraction is another factor of ten smaller, i.e. only a few uL/min. Positive displacement differential pumping is not an easily performed option when one stream has a flow rate that may be hundreds of times larger than the other. In such cases the usual approach is to divide the flow by a tee or Y into two channels which have different lengths and or different flow areas. Thus, for the same pressure drop across both channels the flow rate in the narrower and/or longer channel will be smaller than in the larger one. To divide a one ml/min flow rate so that one channel will carry only 10 or so uL/min, the flow area of its channel must be so small that to prevent partial or even total plugging becomes a serious problem. Moreover, small differences in temperature between the two channels can make substantial differences in liquid viscosity giving rise to changes in the split ratio. Seemingly appropriate flow splitters are available but they are expensive and not entirely dependable unless extreme care is taken to maintain conditions in both legs very constant for long periods of time, not an easy task. For these any other reasons such flow-splitting has not been a very satisfactory option.
One of the most widely used "fixes" for high flow rates is to provide a "pneumatic assist" to the electrostatic forces that are responsible for the dispersion of sample liquid in "pure" electrospray Thus, an annular flow of high velocity gas surrounds the sample liquid emerging from the injection needle and helps in the nebulization. This pneumatically assisted electrospray was tried by Dole et al in their pioneering experiments (references cited) but did not increase the apparent ion current and so was abandoned. A. D. Bruins, L. O. G Weidolf and J. D. Henion [Anal. Chem. 59, 2647 (1987); J. D. Henion, T. R. Covey, A. P. Bruins [U.S. Pat. No. 4,861,988] were the first to show that this pneumatic assist did make possible the electrospray dispersion of sample liquids flowing at rates up to one ml/min. They called the combination "IonSpray" a term which became a trade name for a commercially available system. Another approach, developed by C. M. Whitehouse, S. Shen and J. B. Fenn [U.S. Pat. 5,306,412], is to use mechanical vibration of the injection needle at ultrasonic frequencies to help disperse the liquid. Both of these methods are able to produce charged droplets at high flow rates with liquids that are difficult to spray, but in both cases the analytical sensitivity (MS Signal) tends to be substantially less than pure ES at low flow rates can provide for the same solution. The reason is that the increase in flow rate that these "assists" allow is not accompanied by a concomitant increase in spray current. Consequently, the charge/mass ratio of the initial droplets decreases as flow rate increases so that a smaller fraction of the analyte molecules is transformed into ions and the selected ion current (MS signal) is decreased for each species. In many cases the decrease in MS signal can be tolerated so that aerodynamic or pneumatic assistance of ES dispersion, which is somewhat simpler and more rugged than ultrasonic mechanical vibration, has become widely used. Although these aerodynamic or mechanical "assists" can often produce useful sprays with liquids having high surface tension and conductivity, for particularly refractory liquids they are sometimes used in conjunction with the sheath flow of an "ES friendly" liquid as described above. To be remembered is that in either of these high flow methods all or most of the LC effluent is dispersed as a spray but only a small portion of that effluent evaporates completely and passes into the vacuum system. Therefore, provision must be made to remove the remainder of the liquid and/or pneumatic gas from the spray chamber. Because all the LC effluent is dispersed in the spray chamber the peak fractions cannot very well be recovered intact as cam be done with an an effective splitter.